Nucleic Acids Research Advance Access originally published online on April 22, 2007
Nucleic Acids Research 2007 35(10):3238-3251; doi:10.1093/nar/gkm215
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Nucleic Acids Research, 2007, Vol. 35, No. 10 3238-3251
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Expression of catalytic mutants of the mtDNA helicase Twinkle and polymerase POLG causes distinct replication stalling phenotypes
1Institute of Medical Technology and Tampere University Hospital, Tampere, Finland and 2MRC-Dunn Human Nutrition Unit, Wellcome Trust-MRC Building, Cambridge, UK
*To whom correspondence should be addressed: Tel: +358 3 35518598; Fax: +358 3 35517710; Email: hans.spelbrink{at}uta.fi
Received December 8, 2006. Revised March 6, 2007. Accepted March 27, 2007.
The mechanism of mitochondrial DNA replication is a subject of intense debate. One model proposes a strand-asynchronous replication in which both strands of the circular genome are replicated semi-independently while the other model proposes both a bidirectional coupled leading- and lagging-strand synthesis mode and a unidirectional mode in which the lagging-strand is initially laid-down as RNA by an unknown mechanism (RITOLS mode). Both the strand-asynchronous and RITOLS model have in common a delayed synthesis of the DNA-lagging strand. Mitochondrial DNA is replicated by a limited set of proteins including DNA polymerase gamma (POLG) and the helicase Twinkle. Here, we report the effects of expression of various catalytically deficient mutants of POLG1 and Twinkle in human cell culture. Both groups of mutants reduced mitochondrial DNA copy number by severe replication stalling. However, the analysis showed that while induction of POLG1 mutants still displayed delayed lagging-strand synthesis, Twinkle-induced stalling resulted in maturated, essentially fully double-stranded DNA intermediates. In the latter case, limited inhibition of POLG with dideoxycytidine restored the delay between leading- and lagging-strand synthesis. The observed cause-effect relationship suggests that Twinkle-induced stalling increases lagging-strand initiation events and/or maturation mimicking conventional strand-coupled replication.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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